The goal of this project is to generate a novel PEGylated bispecific, bivalent single chain (scFv) antibody (MAb) construct as part of a new pretargeting delivery strategy that will greatly increase the therapeutic index of radioimmunotherapy (RIT) in metastatic prostate cancer. The underlying hypothesis is that metastatic prostate cancer can be cured without marrow support by systemically targeted radiation therapy as part of combined modality therapy, if the therapeutic index of RIT is increased by a factor of 10. In a Phase I RIT study in patients with androgen-independent metastatic prostate cancer, we have induced therapeutic response using Yttrium-90 (90Y) MAb against an epithelial mucin (MUC-1), but marrow support was needed for effective therapy. In contrast, the strategy for pretargeted RIT (pRIT) disassociates the phase of accumulation of the nonradioactive construct at the sites of prostate cancer from the phase for delivery of the small radioisotope source, thereby dramatically decreasing radiation to normal tissues while preserving the radiation to the cancer. Key issues are addressed for pretargeted therapy, by providing a less immunogenic, modular, specific tumor targeting molecule, and a multivalent radioactive source that will, on subsequent injection, quickly bind and cross link the pretargeted molecules on tumor cells with high avidity. If needed, further strategies have been developed to negate interference from pretargeting molecules that remain in the circulation. Specific Aims have included: 1) select scFv from MUC-1 immune scFv libraries that specifically bind prostate cancer cells using tissue arrays of >300 prostate cancers and an extensive panel of normal tissues; 2) produce an optimized anti-cancedanti-DOTA scFv conjugated PEG ((scFv)4-PEG)); 3) develop an optimized multivalent Y-90 DOTA structure to bind and cross link on tumor, utilizing molecular modeling and biosensor affinity; 4) study the dose/time characteristics of the prostate cancer pretargeting conjugate as In-111 DOTA-(scFv)4-PEG and the multivalent Y-90 DOTA in in vitro and in vivo pharmacokinetic studies to maximize tumor uptake in nude mice bearing human prostate cancers; 5) if needed, implement strategy to negate interference of residual pretargeting molecules in blood; 6) develop protocols and GMP pharmaceuticals for pharmacokinetic studies in prostate cancer patients to be initiated by the next grant period. The investigative team has: the requisite experience, methods and resources. We have made vector modifications allowing site specific scFv conjugation; demonstrated MUC-1 target expression patterns correlating to malignancy and Gleason grade by MAb; selected scFv that selectively stain prostate cancer on tissue arrays. This proposal has a high likelihood of success.